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From 2012.igem.org

Week 4 (07.09-07.15): Make our own biobrick parts

July 09

Prepare liquid SOB medium for co-transformation.

July 10

Amplify glycerol stock bacteria FB (DH5α) on plates.

July 11

1. Identify FA and FB with PCR.

2. Amplify FB-7-10-plate bacteria in liquid LB medium.

July 12

1. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FB-7-11 Concentration: 15.4 ng/ul A260/280: 1.88

2. Amplify FB-7-10-plate1, FB-7-10-plate2, FA-6.29-liq (DH5α), FA-lix and FB-lix bacteria for minipreparing plasmids later.

3. Identify FB with PCR again.We found there were problems with primers used in those reactions, thus leading to the failure.

4. Ligation: D0 and pSB1C3, purified at July 08.

July 13

1. Transformation:

No.name time of heat shock/s

1 D0-FA-FB 90

2 FA-FB 90

3 D0-FA-FB 30

4 FA-FB 30

5 ligation product 90

6 D0 90

2. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: FA-7-13-1-1 Concentration: 15.8 ng/ul A260/280: 1.89

Name: FA-7-13-1-2 Concentration: 10.8 ng/ul A260/280: 2.06

Name: FA-7-13-1-3 Concentration: 13.6 ng/ul A260/280: 2.30

Name: FA-7-13-1-4 Concentration: 12.3 ng/ul A260/280: 2.13

Name: FA-7-13-2-1 Concentration: 9.4 ng/ul A260/280: 2.15

Name: FA-7-13-2-2 Concentration: 12.4 ng/ul A260/280: 1.94

Name: FA-7-13-2-3 Concentration: 20.1 ng/ul A260/280: 1.94

Name: FA-7-13-3-1 Concentration: 11.1 ng/ul A260/280: 2.23

Name: FA-7-13-lix Concentration: 13.3 ng/ul A260/280: 2.08

Name: FB-7-13-yy Concentration: 26.4 ng/ul A260/280: 1.84

July 14

1. Co-transformation:

No.name time of heat shock/s

1 D0-FA-FB 90

2 D0-FA-FB 30

Use SOC liquid medium.

2. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: FB-7-14-1 Concentration: 42.9 ng/ul A260/280: 0.67

Name: FB-7-14-2 Concentration: 21.0 ng/ul A260/280: 2.69

Name: FB-7-14-3 Concentration: 44.7 ng/ul A260/280: 0.59

Name: FB-7-14-4 Concentration: 14.4 ng/ul A260/280: 2.09

Name: FB-7-14-5 Concentration: 33.1 ng/ul A260/280: 0.68

Name: FB-7-14-6 Concentration: 26.1 ng/ul A260/280: 1.64

3. Amplify glycerol stock bacteria co-transformation (BL21*(DE3)) on 3 resistance plates, glycerol stock bacteria D0 on Amp plate.

4. Amplify FB-7-12-2, FB-7-12-4, FA-7-12-2-2 and FA-7-12-2-3 in LB liquid medium, named FB-7-14-2, FB-7-14-4, FA-7-14-2-2 and FA-7-14-2-3.

5. Digest FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with Pst1.

Maybe the concentration is too low that we can't recognize it.

July 15

1. Transformation:

No. name time of heat shock/s Strain

1 D0-FA-FB 90 DH10β

2 FA-FB 90 DH10β

3 D0-FA-FB 30 BL21*(DE3)

4 FA-FB 30 BL21*(DE3)

5 D0-pSB1C3 90 BL21*(DE3)

6 D0 90 BL21*(DE3)

2. Amplify bacteria in LB liquid medium:

No. Name From quantity

1 Co-7-15 co-transformation (BL21*(DE3)) 6

2 D0-7-15 D0-7-14 1

3 Co3-7-15-1-3-1 Co3-7-14-1 1

4 Co3-7-15-1-3-2 Co3-7-14-2 1